Search

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.

The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery larger yeast have one ATF2 gene, while S. bayanus has no similar gene.

We used a plasmid carrying a sequence for autonomous maintenance in Aspergillus (AMA1) and the E. coli uidA gene as a reporter gene to search the A. oryzae and A. niger genomes for DNA fragments having strong promoter activity. Beta-glucuronidase (GUS)-producing A. oryzae transformants containing the No. 8AN derived from A. niger, or the No. 9AO derived from A. oryzae, were constitutive for the expression of the uidA gene when cultivated in the presence of a variety of carbon and nitrogen sources. When the GUS-producing transformants were grown in liquid culture, the No. 8AN showed an increase of approximately 3-fold in GUS activity compared to the amyB (alpha-amylase encoding gene) promoter. There was also a corresponding increase in the amount of GUS gene-specific mRNA. When these transformants were grown as rice-koji, the No. 8AN showed an increase of approximately 6-fold compared to the amyB promoter, and the amount of GUS protein produced also increased. These strong promoter regions might be applicable to the production of other heterologous proteins in Aspergillus species.

Ten standard strains of hiochi bacteria were selected based on the SDS-PAGE patterns of their cellular proteins. We then obtained ten hybridoma systems that secreted highly reactive monoclonal antibodies (MAbs) to the whole cells of each strain. It was apparent that these MAbs were highly reactive and specific for hiochi bacterial whole cells when using an enzyme-linked immunosorbent assay (ELISA). Three MAbs (two against the homo-fermentative hiochi lactobacilli and an MAb against the hetero-fermentative true hiochi bacilli) showed cross-reactivity to some of the other strains of lactobacilli tested. However, the other MAbs did not react with strains other than the immunogen. A sensitive ELISA method for the detection of hiochi bacteria was examined. It was possible to detect the order of 103 cells of the ten standard strains. Using this procedure and a mixture of the ten MAbs, a detection limit of 104 cells or less could be obtained for 98.2% of the hiochi bacteria isolated from sake brewing factories. ...

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per micrograms of integrative vector and 100 colonies per micrograms of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.

Alcohol acetyltransferase (AATase) catalyzes the esterification of isoamyl alcohol by acetyl coenzyme A. The enzyme was solubilized from the microsomal fraction of Saccharomyces cerevisiae Kyokai No. 7, using Triton X-100 and then purified by a series of chromatographic separations: Poly Buffer Exchanger 94 (PBE94), DEAE Toyopearl, Toyopearl HW60, hydroxyapatite, Octyl-Sepharose CL-4B, and hexanol-affinity column chromatography. When the solubilized enzyme was put on PBE94, two active fractions were obtained. The enzyme obtained after affinity column chromatography had a single band on an SDS polyacrylamide gel, and its molecular mass was estimated to be 60 kDa. The enzyme was most active at pH 8.0 and 25 degrees C. It was stable between pH 7.5 and 8.5, but was unstable at temperatures above 10 degrees C. The activity was markedly inhibited by heavy metal ions such as Cd2+, Cu2+, Zn2+, and Hg2+, and sulfhydryl reagents. The Km for acetyl-CoA was 0.19 mM. The internal peptide sequences were also identified.

The whole cellular proteins of various hiochi bacteria were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the electrophoretic patterns, the hiochi bacteria were generally divided into four distinguishable groups, agreeing with the conventional groupings based on physiological and biochemical characters; homo- and hetero-fermentative true hiochi bacilli and homo- and hetero-fermentative hiochi lactobacilli. The SDS-PAGE patterns of the other lactobacilli were clearly different from that of the hiochi bacteria. The SDS-PAGE patterns of SDS-extracted protein from hiochi bacteria can be used as a simple and a useful tool for identification of the microorganisms causing putrefaction of sake.

現在の清酒に比べグルタチオン含有量の高い清酒を開発するため,液胞生理機能欠損変異酵母を用い検討を行った. (1) 液胞生理機能欠損変異酵母において,グルタチオンを菌体外に漏出することが認められた. (2) 液胞生理機能欠損変異酵母に,醸造適性を付与することによりアルコール16%,グルタチオン20ppm以上の清酒を製造することが可能となった. (3) さらに,酵素剤を併用し仕込方法を改良した結果,香り,酒質など品質面において,従来の製品と同等の清酒が製造可能となった. 終わりに臨み,液胞生理機能欠損変異酵母を分譲していただいた東京大学大隅良典教授,国税庁醸造試験所北本勝ひこ博士,ならびに終始ご指導を賜りました弊社常務取締役総合研究所所長布川弥太郎博士に深く感謝いたします.

An autonomously replicating plasmid is expected to increase the frequency of Aspergillus transformation. To construct this type of plasmid, we developed a rapid method of re-isolating autonomously replicating plasmids from Aspergillus transformants. Transformants grown in MM medium under selective pressure for 1-2 days were converted to protoplasts with a cell wall lytic enzyme (e.g. Yatalase). The protoplasts were lysed with phenol/chloroform followed by precipitation with ethanol. The total DNA was treated with RNaseA, re-precipitated with PEG, and then used to transform E. coli. These re-isolated plasmids were mainly the plasmid monomer.

瓶容器での清酒の常温無菌充填について検討し, 次の結果を得た。1. 空中からの火落菌の混入する確率は極めて低いが, 充填機周辺はクリーンルーム乃至クリーンブースの採用が適当と考えられる。2. 熱水 (90℃, 15分間) CIP条件で充填ラインの無菌化が可能であったが, 洗浄面から熱アルカリ工程を入れる必要があった。3.大量充填試験により, 本装置で常温無菌充填が可能であることが分った。4. 熱交換法の代わりに超精密炉過法を行いることにより, 生酒の無菌充填が可能であることが確められた。